Novel Recombinant BCG Tuberculosis Vaccine Designed to Elicit Immune Responses to Mycobacterium Tuberculosis in all Physiological Stages of Infection and Disease

ABSTRACT

A vaccine against  Mycobacteria tuberculosis  (Mtb) is provided. The vaccine comprises a recombinant Bacille Calmette-Guerin (BCG) subunit-based vaccine in which one or more Mtb antigens and one or more Mtb resuscitation or reactivation antigens are overexpressed, and in which at least a portion of the DosR regulon is up-regulated. The vaccine is protective against active Mtb infection both pre- and post-exposure to Mtb, and thus prevents disease symptoms due to the recurrence of a latent Mtb infection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to improved Mycobacterium tuberculosis(Mtb) vaccines that are successful in preventing the development ofsymptoms of tuberculosis, both pre- and post-exposure to Mtb. Inparticular, the invention provides an improved recombinant BacilleCalmette-Guerin (BCG) subunit-based vaccine in which one or more Mtbantigens and one or more Mtb resuscitation or reactivation antigens areoverexpressed, and in which at least a portion of the DosR regulon isup-regulated.

2. Background of the Invention

The current prophylactic (pre-exposure) Mycobacterium tuberculosis (Mtb)vaccine Mycobacterium bovis (M. bovis) BCG, introduced over 60 yearsago, efficiently protects against severe disease manifestation inchildren but fails to prevent the establishment of latent TB or diseasereactivation of infection in adolescents and adults. Moreover,essentially all novel Mtb vaccines currently in clinical trials aredesigned as prophylactic rather than both prophylactic and therapeutic(post exposure) vaccines.

It is believed that Mtb progresses through a series of stages during itsinfectious cycle in man as a reaction to human immune responses and thateach stage is orchestrated by a distinct genetic program which directsthe expression of stage-specific antigens. If this concept is valid,then a truly comprehensive tuberculosis vaccine should include antigensrepresenting each stage as well as antigens that are stage-independent.Latent tuberculosis (LTBI/latency) appears to be one such stage andcurrent evidence suggests that Mtb adopts a unique physiologicalphenotype during latency characterized by bacteriostasis(non-replicating persistence), a switch from aerobic to anaerobicrespiration, expression of the α-crystalline small chaperone protein(Acr/HspX) and increased resistance to several mycobacterialantibiotics.

Maintenance of the non-replicating persistence state, believed to betypical of Mtb in latent lesions, appears to depend on the continuousproduction of Th1 cytokines (IFNγ, IL-12 and TNFα) and nitric oxide andthe localization of MTB within stable granulomas. However, thereactivation of latent Mtb infection, characterized by resumption ofbacterial replication, inflammation and cavitation, can be promptlyprecipitated by immunosuppressive regimens (e.g., corticosterioids orTNFα-antagonist) and occurs in 5-10% of latently infected individuals,perhaps due to acquired tolerance to environmental mycobacteria, age,and, more significantly, HIV disease. This common clinical scenario andthe proven role of the cellular immune system for the maintenance oflatency lead to the conclusion that non-replicating persistence is ametastable phenotype determined by three interacting processes:bacterial replication within latent lesions is constrained by effectorsof the cellular immune system; bacteria within latent lesions monitorthe production of immune effectors; and, decreased production of immuneeffectors results in resumption of replication.

To date, no successful vaccines have been developed which conferimmunity to infection by Mtb and at the same time treat or prevent thedevelopment of symptoms of TB after exposure to Mtb, or as a result ofreactivation of latent infection. A recombinant BCG vaccine, engineeredto elicit an immune response of this kind, might reduce reactivationrates in persons with subtle degrees of immunosuppression produced, forexample, by senescence, diabetes, HIV disease, acquired tolerance toenvironmental mycobacteria or malnutrition. There is thus an ongoingneed to develop new TB vaccines, and it would be particularly useful todevelop a vaccine that can be used both prophylactically and forpost-exposure treatment.

SUMMARY OF THE INVENTION

The present invention is based on the development of a novel recombinantBacille Calmette-Guerin (rBCG) for use as a vaccine. The vaccine may beused prophylactically to prevent Mtb infection in naive individuals.However, the vaccine is also effective for treating individuals who havealready been exposed to and/or infected by Mtb. The vaccine prevents theestablishment of infection and likewise prevents the reactivation oflatent Mtb in individuals that have been previously infected. The rBCGthat is used in the vaccine preparations is genetically engineered toexpress “classical” Mtb antigens and antigens that are relevant toseveral stages of the Mtb life cycle, e.g. latency, reactivation andresuscitation. Thus, the immune response that is generated as a resultof immunization with the vaccine protects the vaccinated individual fromdeveloping an active Mtb infection at any and all stages of exposure toMtb. In particular, the rBCG overexpresses 1) one or more genes encodingMycobacterium tuberculosis (Mtb) antigens that are known to elicitpotent, protective immune responses to Mtb; and 2) one or more genesencoding at least one Mtb resuscitation or reactivation antigen. Theantigen encoding sequences are located on an extrachromosomal element orare integrated into the chromosome of the recombinant BCG. In addition,expression of all or part of the Dos R regulon is up-regulated in thenovel rBCG. The embodiment of the invention in which the antigenencoding sequences are integrated into the chromosome is depictedschematically in FIG. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the rBCG of the invention.

FIG. 2. Diagram of integration plasmid and expression cassette.

FIG. 3A-D. Immunoblots demonstrating the expression of chromosomallyintegrated antigen cassettes. A, Expression or Rv0867c from AERAS-407precursor AFV-102pRC108, Lane 1=AFV-102pRC108, Lane 2=AFV-102; B,Overexpression of Ag85A and Ag85B from AERAS-407 precursor AFV-102RC108,Lane 1=AFV-102, Lane 2=AFV-102pRC108; C, Expression of Rv3407 fromAERAS-407 precursor AFV-102pRC108, Lane 1=AFV-102, Lane 2=AFV-102pRC108;D, Expression of DosR (Rv3133c) from AERAS-407 precursor AFV-102pRC108,Lane 1=AFV-102, Lane 2=AFV-102pRC108.

FIG. 4. Diagram of a second integration plasmid and expression cassette.

FIGS. 5A and B. Rv3804c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 6A and B. Rv1886c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 7A and B. Rv0867c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 8A and B. Rv1009c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 9A and B. Rv1884c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 10A and B. Rv2389c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 11A and B. Rv2450c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 12A and B. Rv2623c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 13A and B. Rv0288c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 14A and B. Rv2626c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 15A and B. Rv2005c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 16A and B. Rv1996c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 17A and B. Rv0685c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 18A and B. Rv0824c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 19A and B. Rv2029c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 20A and B. Rv2627c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 21A and B. Rv2744c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 22A and B. Rv3347c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 23A and B. Rv1130c amino acid sequence (A) and nucleotide sequence(B).

FIGS. 24A and B. Rv1169c amino acid sequence (A) and nucleotide sequence(B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a novel rBCG for use in Mtb vaccinepreparations. The rBCG is genetically engineered to overexpress 1) oneor more Mtb antigens, which may include so-called “classical” Mtbantigens such as Rv1886c-Ag85B (“85B”) and Rv3804c-Ag85A (“85A”), amongothers; and 2) at least one Mtb resuscitation/reactivation antigen. Inaddition, in the rBCG of the invention, the DosR regulon, or a portionthereof, is up-regulated. The DosR regulon, comprised of 48 genes, istypically activated in Mtb as a result of a post-infection drop in O₂tension (hypoxia).

The Mtb antigens that are selected for inclusion in the rBCG of theinvention are generally those which are known or predicted to elicit aprotective immune response in individuals exposed to the antigens.Various criteria may be used to select suitable antigens, including butnot limited to: the observation that individuals with immune responsesto the antigen are able to control Mtb infection, determination that theantigen contains one or more T-cell epitopes via an immunoinformaticsanalysis (e.g. using a program such as that which is found at thewebsite of the Technical University of Denmark:cbs.dtu.dk/services/NetCTL, in which the analysis is based on theidentification of proteosome cleavage sites, endoplasmic reticulum (ER)transport efficiency, major histocompatibility (MHC) class 1 bindingaffinity, etc.); an analysis of experimental evidence based on anin-depth literature search; sorting or ranking of results according tovarious parameters such as macrophage survival/persistence;up-regulation of expression by the two-component system MprAB; responseto hypoxia; involvement in dormancy; expression in lung tissue; geneswith promoters that are co-regulated with Rv2031 (Acr); proteins thatare secreted (which are more accessible to the immune system); presenceof repeats (many virulence associated proteins have amino acid repeatdomains); ability to act as a B-cell immunogen; cell wall associated orcell walls biogenesis (membrane-exposed/associated proteins areconsidered to be more accessible to the immune system; existing vaccineefficacy data; uniqueness to Mtb; etc.) Thus, antigens may be selectedbased on experimental data demonstrating their efficacy, oralternatively (or in addition) such antigens may be selected based ontheir predicted abilities. Those of skill in the art are familiar withthe implementation of such analyses, and with developing scoring orranking systems in order to assign a weighted comparative score tocandidate antigens. For example, numeric scores may be assigned for eachattribute under consideration, and the antigens with the highestcumulative totals may be selected for use. In addition, otherconsiderations may play a role in the decision making process, some ofwhich are practical in nature (e.g. the availability of the antigen, theease of expressing the antigen, the ease of measuring immune responsesto the antigen, etc.)

The starting pool of antigens may be any or all known Mtb open readingframe (ORF) products. Those of skill in the art are familiar withsources for identifying Mtb ORF products, e.g. the “Tuberculist” websitesponsored by the Pasteur Institute. In one embodiment of the invention,the antigens from which a selection may be made include but are notlimited to all ORF products of M. tuberculosis H37Rv as identified inGenBank Accession #AL123456 (NC 000962).

In a preferred embodiment of the invention, the antigens that areexpressed by the rBCG are selected from but are not limited to aninitial group of 189 antigens presented in Table 1.

TABLE 1 List of 189 selected antigens No. Gene (name) Size NCBIAnnotation [Updated annotation]^((a)) Rv0079 273 hypothetical proteinRv0101 nrp 2512 probable peptide synthase (nrp) Rv0125 pepA 355 probableserine protease pepA (MTB32A) [DegQ] Rv0170 mce1B 346 MCE-family proteinmce1B [Ttg2C, periplasmic component ABC transporter] Rv0198c 663possible zinc metalloprotease [PepO, predicted metalloendopeptidase]Rv0211 pckA 606 phosphoenolpyruvate carboxykinase pckA (GTP) Rv0227c 421probable conserved membrane protein Rv0243 fadA2 440 acetyl-coAacetyltransferase [beta keto-thiolase] Rv0251c hsp [acr2] 159 heat-shockprotein hsp (heat-stress induced ribosome-binding protein A) Rv0282 631hypothetical protein [AAA ATPase] Rv0283 538 possible conserved membraneprotein [ATP/GTP-binding protein] Rv0284 [ftsk] 1330 possible conservedmembrane protein [chromosome partition ATPase] Rv0285 PE5 102 PE familyprotein (PE5) Rv0286 PPE4 513 PPE family protein (PPE4) Rv0287 exsG 97ESAT-6-like protein esxG (conserved hypothetical protein TB9.8) Rv0288esxH (TB10.4) 96 low MW protein antigen 7 esxH (10 kDa antigen) CFP-7,TB10.4) Rv0289 295 hypothetical protein [transporter] Rv0290 472probable conserved trans-membrane protein (mgcP3) [transporter] Rv0292331 probable conserved trans-membrane protein Rv0350 dnaK 625 molecularchaperone DnaK Rv0351 grpE 235 probable grpE protein (HSP-70 cofactor)Rv0383c 284 possible conserved secreted protein Rv0384c clpB 848probable endopeptidase ATP binding protein ClpB (chain B) heat-shockprotein F84.1 Rv0450c mmpl4 967 probable conserved trans-membrane mmpL4[drug exporting] Rv0467 icl [aceA] 428 isocitrate lyase (icl) Rv0468fadB2 268 3-hydroxybutyryl-CoA dehydrogenase Rv0503c cmaA2 302cyclopropane-fatty-acyl-phospholipid-synthase 2 (cyclopropane mycolicacid synthase 2, CMAA2) Rv0569 88 hypothetical protein Rv0572c 113hypothetical protein Rv0574c [pgsA] 380 hypothetical protein [pgsApoly-gamma-glytamyl biosyntehsis] Rv0588 yrbE2B 295 conservedhypothetical integral membrane protein YrbE2B [putative Ttg2B, ABC-typetransport system involved in resistance to organic solvents, permeasecomponent) Rv0628c 383 hypothetical protein Rv0685 Tuf 396 elongationfactor Tu, tuf [iron-regulated] Rv0754 PE_PGRS11 584 PE-PGRS familyprotein (PE_PGRS11) [phosphoglycerate mutase] Rv0798c cfp29 265 29 kDaantigen CFP-29 [linocin M-18 bacteriocin] Rv0824c desA1 389 probableacyl-[-acyl-carrier-desaturase desA1] Rv0847 lpqS 130 probablelipoprotein LPQS Rv0867c rpfA 407 possible conserved trans-membraneprotein [transglycosylase, rpfA] Rv0885 340 hypothetical protein Rv1006567 hypothetical protein Rv1009 rpfB 362 possibleresuscitation-promoting factor rpfB [transglycosylase, C5 adhesiondomain] Rv1057 393 hypothetical protein Rv1094 desA2 271 possibleacyl-[-acyl-carrier protein] desaturase (DESA2) Rv1124 ephC 316 probableepoxide hydrolase EPHC (epoxide hydratase) Rv1130 [prpD] 526hypothetical protein [2 methyl-citrate dehydratase] Rv1131 gltA1 393citrate synthase (glaA1) Rv1169c PE11 100 PE family protein (PE11)[triacyl glycerol lipase] Rv1174c [sak5] 110 low MW T-cell antigen TB8.4[secretion antigen SA5K] Rv1182 papA3 472 probable conserved polyketidesynthase associated protein PAPA3 Rv1186c 538 hypothetical protein[regulator of polyketide synthase expression] Rv1187 rocA 543 probableproline-5-carboxylate dehydrogenase rocA Rv1188 329 probable prolinedehydrogenase Rv1196 PPE18 391 probable proline dehydrogenase PPE familyprotein (mtb39a) [MTB39a] Rv1221 sigE 257 RNA polymerase sigma-70 factor(SigE) Rv1347c 210 hypothetical protein [GCN5-related N-acetyltransferase fold] Rv1348 [lrtA] 859 probable drug-transporttrans-membrane ATP-binding protein ABC-transporter [MdlA/MsbA essentialABC transporter, siderophore interaction protein] Rv1349 [irtB] 579possible drug-transport trans-membrane ATP-binding proteinABC-transporter [ATM1 ABC siderophore- iron transporter] Rv1411c lprG236 possible conserved lipoprotein lprG Rv1436 gap 339glyceraldehyde-3-phosphate dehydrogenase Rv1461 [sufB] 846 hypotheticalprotein [sufB, cytosine desulfurase activator] Rv1462 [sufD] 397hypothetical protein [sufD, cytosine desulfurase activator] Rv1464 csd[sufS] 417 possible cysteine desulfurase csd [SufS] Rv1465 [nifU] 162possible nitrogen fixation related protein [IscU] Rv1466 115hypothetical protein [PaaD, predicted metal-sulfur cluster biosyntheticenzyme] Rv1477 ripA 427 hypothetical invasion protein [Nlp_p60 cell-wallhydrolase] Rv1478 241 hypothetical invasion protein [Nlp_p60 cell-wallhydrolase] Rv1594 nadA 349 quinoline synthetase (nadA) Rv1636 TB15.3 146iron-regulated conserved hypothetical protein TB15.3 [USP] Rv1733c 210probable conserved transmembrane protein Rv1734c 80 hypothetical protein[dihydrolysine acetyl-transferase] Rv1735c 165 hypothetical membraneprotein Rv1736c narX 652 possible nitrate reductase narX Rv1737c narK2395 possible nitrate/nitrite transporter narK2 Rv1738 94 hypotheticalprotein Rv1793 esxN 94 putative ESAT-6-like protein ESXN (ESAT-6-likeprotein 5) Rv1812c [ndH] 400 possible dehydrogenase [Ndh, NADHdehydrogenase, FAD-containing subunit] Rv1813c 143 hypothetical proteinRv1876 bfrA 159 probable bacterioferritin bfrA Rv1884c rpfC 176 probableresuscitation-promoting factor rpfC [transglycosylase] Rv1886c ftpB(Ag85B) 325 secreted antigen 85-B FBPB (85-B) (mycolyl-transferase 85B)Rv1908c katG 740 catalase-peroxidase-peroxinitritase-T katG Rv1926cmpt63 159 immunogenic protein MPT63 (16 kDa immunoprotectiveextracellular antigen) Rv1980c mpb64 228 immunogenic protein MPT64Rv1986 199 probable conserved integral membrane protein [lysine effluxpermease] Rv1996 317 hypothetical protein [USP] Rv1997 ctpF 905 probablemetal cation transporter P-type APTase cptF Rv1998c 258 hypotheticalprotein Rv2004c 498 hypothetical protein [predicted kinase] Rv2005c 295hypothetical protein [USP-like] Rv2006 otsB1 1327 probabletrehalose-6-phosphate phosphatase OSTB1 Rv2007c fdxA 114 probableferrodoxin fdxA Rv2008c 441 hypothetical protein [predicted ATPase]Rv2011c 143 hypothetical protein[transcription regulator] Rv2028c 279hypothetical protein [USP] Rv2029c pfkB 339 possible phosphofructokinase(pfkB) Rv2030c 681 hypothetical protein [putative esterase/transferase]Rv2031c acr 144 heat-shock protein HspX (alpha-crystallin homolog)(α-crystallin) 14 kDa antigen Hsp16.3 Rv2032 acg 331 conservedhypothetical protein Acg Rv2110c [prcB] 291 proteosome (beta subunit)PrcB [HslV protease] Rv2123 PPE37 473 PPE family protein (PPE37) Rv2140cTB18.6 [pepB] 176 hypothetical protein (TB18.6) [PEBP, bacterial/archealphosphatidylethanolamine- binding protein] Rv2182c [pslC] 2471-acylglycerol-3-phosphate O-acyltransferase Rv2224c [caeA] 520 probableexported protease [cae, carboxylase A, TAP-like protein] Rv2244 acpM 115acyl-carrier protein (acpM) Rv2245 kasA 416 3-oxoacyl-(acyl carrierprotein) synthase (kasA) Rv2246 kasB 438 3-oxoacyl-(acyl carrierprotein) synthase (kasB) Rv2251 [glcD] 475 possible flavoprotein [GlcD,FAD/FMN-containing dehydrogenases] Rv2377c mbtH 71 putative conservedprotein MBTH Rv2378c mbtG 431 lysine-N-oxygenase MBTG [lucD] Rv2380cmbtE 1682 peptide synthase MBTE [EntF] Rv2381c mbtD 1004 polyketidesynthase MBTD [acyl-transferase, KR domain] Rv2382c mbtC 444 polyketidesynthase MBTC Rv2383c mbtB 1414 phenyloxazoline synthase (MBTB) [EntF,GrsT] Rv2386c mbtI 450 anthranilate synthase component I (MBTA)[salicylate synthase] Rv2389c rpfD 154 probable resuscitation-promotingfactor rpfE [transglycosylase] Rv2428 ahpC 195 alkyl hydroxyperoxidereductase C protein ahpC Rv2429 ahpD 177 alkyl hydroxyperoxide reductaseD protein ahpD Rv2430c PPE41 194 PPE family protein (PPE41) Rv2450c rpfE172 probable resuscitation-promoting factor rpfE [transglycosylase]Rv2457c clpX 426 ATP-dependent protease ATP-binding subunit (CplX)Rv2466c 207 hypothetical protein [putative DsbA_FrnE] [thioloxidoreductase, polyketide biosynthesis] Rv2510c 533 hypotheticalprotein [ATP binding domain] Rv2515c 415 hypothetical protein [putativezinc peptidase] Rv2516c 250 hypothetical protein Rv2557 224 hypotheticalprotein Rv2590 fadD9 1168 probable fatty-acid coA ligase fadD9 Rv2620c141 probable conserved trans-membrane protein Rv2621c 224 possibletranscriptional regulatory protein Rv2622 273 possible methyltransferase(methylase) Rv2623 TB31.7 297 hypothetical protein TB31.7 [USP] Rv2625c393 probable conserved trans-membrane alanine-rich and leucine-richprotein [zinc-protease M-50 CBS domain] Rv2626c 143 hypothetical protein[CBS pair-binding/regul, euk] Rv2627c 413 hypothetical protein Rv2628120 hypothetical protein Rv2629 374 hypothetical protein [peptide chainrelease factor erF1] Rv2657c 86 probable phiRv2 prophage protein [MerRregulatory protein] Rv2659c 375 probable phiRv2 prophage integraseRv2660 75 hypothetical protein Rv2710 sigB 323 RNA polymerase sigmafactor (SigB) Rv2744c 35kd-Ag [pspA] 270 conserved 35 kDa alanine-richprotein [phage-shock protein IM30] Rv2780 ald 371 secreted L-alaninedehydrogenase ald (40 kDa antigen, TB43) Rv2833c ugpB 436 probableSn-glycerol-3-phosphate-binding lipoprotein UGPB Rv2856 nicT 372possible nickel-transport integral membrane protein nicT Rv2869c 404probable conserved trans-membrane protein [putative pdz membraneassociated zinc-metalloprotease] Rv2875 mpt70 193 major secretedimmunogenic protein MPT70 Rv2930 fadD26 626 fatty-acid-coA ligase FadD26Rv2986c hupB 214 probable DNA-binding protein HU homolog HupB(histone-like protein, 21 kDa laminin-2 binding protein) Rv2999 lppY 321probable conserved lipoprotein LPPY Rv3126c 104 hypothetical proteinRv3127 344 hypothetical protein [possible nitroreductase] Rv3129 110hypothetical protein Rv3130c tgs1 463 hypothetical protein[diacylglycerol acyltransferase] Rv3131 nfnB 332 hypothetical protein[possible nitroreductase NfnB] Rv3132c devS 578 two-component sensorhistidine kinase DevS Rv3133c devR 217 two-component transcriptionregulatory protein DevR Rv3134c 268 hypothetical protein [USP] Rv3139fadE24 468 probable acyl-coA dehydrogenase FadE24 Rv3140 fadE23 401probable acyl-coA dehydrogenase FadE23 Rv3173c 200 probabletranscriptional regulatory protein (TetR/acRR family) Rv3229c desA3 427possible linoleoyl-coA desaturase (delta-(6)-desaturase) Rv3250c rubB495 probable rubredoxin rubB Rv3251c rubA 55 probable rubredoxin rubARv3283 sseA 297 probable thiosulfate sulfurtranserase SSEA (rhodanase)Rv3290c lat 449 L-lysine epsilon aminotransferase Rv3347c PPE55 3157 PPEfamily protein (PE55) [8 copies pentapeptide repeats] Rv3372 ostB2 391possible trehalose-6-phosphate phosphatase (OSTB2) Rv3515c fadD19 548AMP-dependent fatty-acid-coA ligase FadD19 Rv3516 echA19 263 enoyl-coAhydratase/isomerase (echA19) Rv3546 fadA5 391 acetyl-coAacetyltransferase (FadA5) Rv3570c [ncnH] 394 possible oxidoreductase[NcnH, naphthocyclinone hydroxylase] Rv3593 lpqF [penP] 452 probableconserved lipoprotein LPQF [PenP, beta-lactamase class A] Rv3597c lsr2112 probable iron-regulated LSR2 protein precursor Rv3616c [espB] 392conserved hypothetical alanine-rich and glycine-rich protein [ESATsecretion system component] Rv3619c esxV 94 putative ESAT-6-like proteinESXV (ESAT-6-like protein 1) Rv3660c 350 hypothetical protein Rv3763lpqH 159 19 kDa lipoprotein antigen precursor LPQH Rv3804c fbpA (Ag85A)338 secreted antigen 85-A FBPA (85-A) (mycolyl-transferase 85A) Rv3812PE_PGRS62 504 PE-PGRS family protein (PE_PGRS62) Rv3833 263transcriptional regulatory protein [probable araC family] Rv3839 258hypothetical protein Rv3840 137 probable transcriptional regulatoryprotein [cell-envelope related transcription attenuator] Rv3841 bfrB 181possible bacterioferritin bfrB Rv3871 [Ftsk] 591 hypothetical protein[FtsK_SPOIIIE] Rv3873 PPE68 368 PPE family protein [PPE68, RD1 T/Bimmunogen] Rv3874 esxB 100 10 kDa culture filtrate antigen ESXB (LHP,CFP-10) Rv3875 esxA 95 6 kDa early secretory antigenic target ESXA(ESAT-6) Rv3876 [Ftsk] 666 conserved hypothetical proline andalanine-rich protein [chromosome partitioning ATPase] Rv3878 280conserved hypothetical alanine-rich protein Rv3879c 729 hypotheticalalanine and proline-rich protein ^((a))NCBI annotation is based onAccession # AL123456 (NC_000962); updated annotation is based onbioinformatic analyses, data from MTB-related servers and experimentalevidence.

In a more preferred embodiment of the invention, the antigens that areexpressed by the rBCG are the 45 antigens presented in Tables 2 and 3.

TABLE 2 Top-ranking antigens (according to Class/Phase) Gene NCBIannotation Score Score No. (name) Size [Updated annotation]^((a)) QualQuant Class: DORMANCY/DosR Rv1738 94 hypothetical protein 9 14 Rv2623TB31.7 297 heat-shock protein TB31.7 9 14 [universal stress protein]Rv2031c acr 144 heat-shock protein HspX (alpha- 8 14 (α-crystalllin)crystallin homolog) 14 kDa antigen Hsp16.3 Rv2032 acg 331 conservedhypothetical protein Acg 8 13 [nitroreductase] Rv2626c 143 hypotheticalprotein 8 13 [CBS pair-binding/regulation, euk] Rv2005c 295 hypotheticalprotein 8 12 [USP-like] Rv3127 344 hypothetical protein 8 12 [possiblenitroreductase] Rv1733c 210 probable conserved 8 11 trans-membraneprotein Rv1996 317 hypothetical protein [USP] 8 10 Rv2628 120hypothetical protein 8 9 Rv0079 273 hypothetical protein 7 11 Rv3130c[tgs1] 463 hypothetical protein 7 11 [diacylglycerol acyltransferase]Rv3131 [nfnB] 332 hypothetical protein 7 11 [possible nitroreductaseNfnB] Rv1813c 143 hypothetical protein 7 9 Rv2006 otsB1 1327 probabletrehalose-6-phosphate 7 9 phosphatase OTSB1 Rv2029c pfkB 339 possiblephophofructokinase (pfkB) 7 9 Rv2627c 413 hypothetical protein 7 9[serine endopeptidase] Rv2030c 681 hypothetical protein 6 10 [putativeesterase/transferase] Rv3132c devs 578 two component sensor histidine 610 kinase DEVS Rv2629 374 hypothetical protein 6 9 [peptide releasefactor erf-1] Class: RESUSCITATION Rv2450c rpfE 172 probableresuscitation-promoting 9 14 factor rpfE [transglycosylase] Rv1009 rpfB362 possible resuscitation-promoting 9 13 factor rpfB [transglycosylase,C5 adhesion domain] Rv0867c rpfA 407 possible conserved trans-membrane 912 protein [transglycosylase, rpfA] Rv2389c rpfD 154 probableresuscitation-promoting 8 10 factor rpfD [transglycosylase] Rv1884c rpfC176 probable resuscitation-promoting 7 8 factor rpfC [transglycosylase]Class: REACTIVATION Rv1009 rpfB 362 possible resuscitation-promoting 913 factor rpfB [transglycosylase, C5 adhesion domain] Rv0867c rpfA 407possible conserved trans-membrane 9 12 protein [transglycosylase, rpfA]Rv0288 esxH (TB10.4) 96 low MW protein antigen 7 esxH 8 13 (10 kDaantigen) CFP-7, TB10.4) Rv0685 Tuf 396 elongation factor Tu 8 9[iron-regulated] Rv0824c desA1 389 probable acyl[-acyl-carrier- 7 10desaturase desA1] Rv2744c 35kd-Ag 270 conserved 35 kDa alanine-rich 7 8[pspA] protein [phage-shock protein IM30] Rv3347c PPE55 3157 PPE 55Family Protein 6 10 [8 copies pentapeptide repeats] Rv1130 prpD 526hypothetical protein 6 9 [2 methyl-citrate dehydratase] Rv1169c PE11 100PE family protein (PE11) 6 9 [triacyl glycerol lipase] Class: CLASSICALRv1886c fbpB (Ag85B) 325 mycolyl transferase/fibronectin 8 14 bindingRv1980c mpb64 228 antigen MPT64/MPB64 7 13 Rv3804c fbpA (Ag85A) 338mycolyl transferase/fibronectin 7 13 binding Rv3875 esxA 95 6 kDa earlysecretory antigen esxA 6 11 Rv1926c mpt63 159 immunogenic protein MPT636 10 (16 kDa immunoprotective extracellular protein) Rv0467 icl 428isocitrate lyase (icl) [AceA] 6 9 Class: OTHERS Rv3873 PPE68 368hypothetical protein 8 13 Rv1908c katG 740catalase-peroxidase-peroxinitritase-T 7 10 (KATG heme dependent) Rv1174csak5 110 low MW T-cell antigen TB8.4 7 9 (secreted) Rv1349 irtB 579probable drug transport ATP-binding 7 9 protein ABC transporter [ATM1ABC siderophor-iron transporter] Rv2780 ald 371 secreted L-alaninedehydrogenase 7 9 ALD (40 kDa antigen) (TB4.3, cell associated pyridinenucleotide transhydrogenase) Rv2620c 141 probable conservedtransmembrane 7 8 protein Rv1793 esxN 94 putative ESAT-6 like protein 69 (ESXN, ESAT-6 like protein 5) ^((a))NCBI annotation is based onAccession # AL123456 (NC_000962); updated annotation is based onbioinformatic analyses, data from MTB-related servers and experimentalevidence.The list of 45 high-ranking antigens were classified according to thefollowing classes:

-   -   DosR regulon genes (Voskuil et al., 2003, J. Exp Med        198(5):705-713; Voskuil et al. 2004, Tuberculosis 84, 218-227)    -   Resuscitation genes    -   Reactivation genes (essentially according to Talaat et al.,        2007, Proc. Natl. Acad. Sci. 189(21):7877-7886)    -   Classical genes    -   Others (mainly involved in persistence and stress response)        Some of the antigens appear in more than one class in Table 3.

TABLE 3 Top-ranking antigens sorted by qualitative and quantitativescores NCBI Annotation Score Score No. Gene (Name) Size [Updatedannotation]^((a)) (Qual) (Quant) Class/Phase GROUP 1 Rv1738 94hypothetical protein 9 14 DosR Rv2450c rpfE 172 probable resuscitation-9 14 Resuscitation promoting factor rpfE [transglycosylase] Rv2623TB31.7 297 hypothetical protein 9 14 DosR TB31.7 [USP] Rv1009 rpfB 362possible resuscitation- 9 13 Reactivation promoting factor rpfBResuscitation [transglycosylase, C5 adhesion domain] Rv0867c rpfA 407possible conserved 9 12 Reactivation trans-membrane proteinResuscitation [transglycosylase, rpfA] Rv2031c acr 144 heat-shockprotein HspX 8 14 DosR (α-crystallin) (α-crystallin homolog) 14 kDaantigen Hsp 16.3 Rv1886c fbpB 325 secreted antigen 85-B 8 14 Classical(Ag85B) FBPB (85-B) (mycolyl-transferase 85B) Rv0288 esxH 96 low MWprotein antigen 7 8 13 Reactivation (TB10.4) esxH (10 kDa antigen)CFP-7, TB10.4) Rv2032 acg 331 conserved hypothetical 8 13 DosR proteinAcg [nitroreductase] Rv2626c 143 hypothetical protein 8 13 DosR [CBSpair- binding/regulation, euk] Rv3873 PPE68 368 PPE family protein[PPE68, 8 13 Others RD1 T/B immunogen] Rv2005c 295 hypothetical protein8 12 DosR [USP-like] Rv3127 344 hypothetical protein 8 12 DosR [possiblenitroreductase] GROUP II Rv1733c 210 probable conserved 8 11 DosRtrans-membrane protein Rv1996 317 hypothetical protein 8 10 DosR [USP]Rv2389c rpfD 154 probable resuscitation- 8 10 Resuscitation promotingfactor rpfD [transglycosylase] Rv0685 Tuf 396 elongation factor Tu 8 9Reactivation [iron-regulated] Rv2628 120 hypothetical protein 8 9 DosRRv1980c mpb64 228 immunogenic protein 7 13 Classical MPT64 Rv3804c fbpA338 secreted antigen 85-A 7 13 Classical (Ag85A) FBPA (85-A)(mycolyl-transferase 85A) Rv0079 273 hypothetical protein 7 11 DosRRv3130c [tgs1] 463 hypothetical protein 7 11 DosR [diacylglycerolacyltransferase] Rv3131 [nfnB] 332 hypothetical protein 7 11 DosR[possible nitroreductase NfnB] Rv0824c desA1 389 probableacyl[-acyl-carrier- 7 10 Reactivation desaturase desA1] Rv1908c katG 740catalase-peroxidase- 7 10 Others peroxinitritase-T katG Rv1174c [sak5]110 Low Mw T-cell antigen 7 9 Others TB8.4 [secretion antigen SA5K]Rv1349 [irtB] 579 probable drug transport 7 9 Others ATP-binding proteinABC transporter [ATM1 ABC siderophore-iron transporter] Rv1813c 143hypothetical protein 7 9 DosR Rv2006 otsB1 1327 probable trehalose-6- 79 DosR phosphate phosphatase OSTB1 Rv2029c pfkB 339 possible 7 9 DosRphosphofructokinase (pfkB) Rv2627c 413 hypothetical protein 7 9 DosR[serine endopeptidase] Rv2780 ald 371 secreted L-alanine 7 9 Othersdehydrogenase ald (40 kDa antigen, TB43) GROUP III Rv1884c rpfC 176probable resuscitation- 7 8 Resuscitation promoting factor rpfC[transglycosylase] Rv2620c 141 probable conserved 7 8 Otherstransmembrane protein Rv2744c 35kd-Ag 270 conserved 35 kDa alanine- 7 8Reactivation [pspA] rich protein [phage-shock protein IM30] Rv3875 esxA95 6 kDA early secretory 6 11 Classical antigenic target ESXA (ESAT-6)Rv1926c mpt63 159 immunogenic protein 6 10 Classical MPT63 (16 kDaimmunoprotective extracellular protein) Rv2030c 681 hypothetical protein6 10 DosR [putative esterase/transferase] Rv3132c devs 578 two componentsensor 6 10 DosR histidine kinase DEVS Rv3347c PPE55 3157 PPE familyprotein (PE55) 6 10 Reactivation [8 copies pentapeptide repeats] Rv0467icl 428 isocitrate lyase(icl) [AceA] 6 9 Classical Rv1130 [prpD] 526hypothetical protein 6 9 Reactivation [2 methyl-citrate dehydratase]Rv1169c PE11 100 PE family protein (PE11) 6 9 Reactivation [triacylglycerol lipase] Rv1793 esxN 94 putative ESAT-6-like 6 9 Others proteinESXN (ESAT-6-like protein 5) Rv2629 374 hypothetical protein 6 9 DosR[peptide release factor erF1] ^((a))NCBI annotation is based onAccession # AL123456 (NC_000962); updated annotation is based onbioinformatic analyses, data from MTB-related servers and experimentalevidence.

The list of 45 high-ranking antigens were sorted by the qualitativescore and then by the quantitative score according to this invention andas discussed in Example 1. This method leads to 3 groups as follows:

Group I: antigens with qualitative scores 9-8, and within the antigenswith a qualitative score of 8, the quantitative score cutoff is 12.Group II: antigens with qualitative scores 8-7, the quantitative scorecutoff of 7 is 9.Group III: antigens with qualitative score 7-6, the quantitative scorecutoff of 6 is 9. Antigens with more than 5 transmembrane segments wereremoved from the list.

In general, at least one such antigen will be overexpressed and severaldifferent antigens may be overexpressed. For example, about 1-20 or moreof such antigens, or alternatively about 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or only 1 of such antigens will beoverexpressed by the rBCG. Further, multiple copies of one or more ofthe antigens may be encoded and overexpressed in the rBCG. The aminoacid sequences of selected antigens, and the nucleic acid sequences thatencode them, are depicted in FIGS. 5A-B to 25A-B.

In addition, the rBCG contains nucleic acid sequences comprising one ormore genes that encodes and overexpresses at least one Mtbresuscitation/reactivation antigen. Those of skill in the art will beaware that the precise definition of “resuscitation” and “reactivation”antigens may vary somewhat within the field, and in some cases, thedefinitions may overlap. For the purposes of the invention, it is notnecessary to separate resuscitation and reactivation antigens as all areidentified as being of significance in the outgrowth of Mtb from thelatent state. In the most accurate sense, resuscitation antigens (i.e.resuscitation promoting factors) are a subset of reactivation antigensdefined as having significant sequence or functional homology to theresuscitation promoting factor of Micrococcus luteus (G Mukamolova etal, Archives of Microbiology, Volume 172, 1999). For the purposes of thepresent application, a “reactivation” antigen is a protein expressed byM. tuberculosis which elicits an immune response in humans with activetuberculosis but not latent Mtb infection. They may also be identifiedas immunogens expressed by Mtb during outgrowth from a non-replicativestationary phase in the Wayne model of latent tuberculosis. This mayinclude molecules that are expressed during the emergence of Mtb fromthe dormant latent state into active tubercle bacilli. Examples ofsuitable reactivation/resuscitation antigens include Rv0867c, Rv0288,Rv1009, Rv0685, Rv0824c, Rv2744c, Rv3347c, Rv1130, Rv1169c, Rv1009,Rv1884c, Rv2389c, and Rv2450c. In a preferred embodiment, thereactivation/resuscitation antigens expressed are Rv0867c, Rv1884c, andRv2389c.

In addition, in the rBCG of the invention the genes of the DosR(Dormancy Survival Regulator) regulon, or a portion thereof, areupregulated and expressed. The entire regulon may be upregulated, or asuitable portion thereof. For example, genes that encode antigens thatare recognized by individuals with latent TB may be the most suitablefor upregulation. Examples of DosR upregulated antigens include Rv1738,Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996,Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c,Rv2030c, Rv3132c, and Rv2629. It is noteworthy that some overlap existsbetween latency and reactivation antigens, possibly reflecting theextended presence of latency related antigens in previously dormantorganisms re-entering an active growth phase or their function in boththe dormant and actively replicating state after reactivation in themammalian host. Antigens that may be considered to overlap betweenlatency and reactivation are listed in Table 4.

TABLE 4 Antigens that overlap latency and reactivation Reactivation/dosRAntigens Rv1996 Rv2005 Rv2029 Rv2623 Rv2626 Rv2727

By “up-regulate” we mean that expression of each of the individual genesof the regulon or their translated proteins is increased above the levelat which they are expressed when the regulon is in a “repressed” state.Proteins of the DosR regulon are normally expressed at a relatively lowlevel. Upon oxygen starvation and/or the presence of oxidative nitrogencompounds the DosS and DosT proteins of TB complex organismsautophosphorylate and transfer this phosphate to DosR. DosR then bindsto discrete sequences upstream of DosR regulated genes therebyactivating their transcription and upregulating this group of genes andgene products which constitute the DosR regulon. Upregulation within thepractice of this invention mimics this oxygen starvation effect wherethe DosR genes have increased transcription.

Those of skill in the art are familiar with approaches to geneticallyengineering an organism in order to up-regulate selected genes ofinterest, or selected regulons of interest. Such approaches include butare not limited to overexpression of the regulator, introduction ofmutations in the regulator or sensor which render them constitutivelyactive, the introduction of regulators which mimic the function of theregulator in question, introduction of kinases or feedback loop productswhich activate the sensor or regulator, or the introduction ofgenes/gene products which mimic the environmental state which causesactivation of the sensor or regulator. In a preferred embodiment, theDosR regulon is up-regulated by over expressing the response regulatorDosR (Rv3133c) of the DosRST “two component” regulatory system.

In another preferred embodiment, the vaccine includes one or more ofRv1908, Rv3873, Rv2780 and Rv1349. These are immunopotent antigens whichwere identified in silico and/or by experimentation.

In addition, the rBCG of the invention may encode antigens selectedbased on other criteria, such as demonstrated protective efficacy in ananimal model or the expression of the antigen by Mtb but not BCG (JMattow et al., Electrophoresis, 22:2936-2946, 2001, P. R. Jungblut,Molecular Microbiology, 33:1103-1117, 1999, H. J. Mollenkopf et al.,Infection and Immunity, 72:6471-6479, 2004). In a preferred embodiment,the rBCG of the invention expresses Rv3407, which is normally expressedby Mtb but not BCG and has been shown to protect against tuberculosis ina mouse model.

The BCG that is genetically engineered as described herein may be of anyBCG strain considered suitable, including but not limited to BCG strainsBCG₁₃₃₁, BCG Pasteur, BCG Tokyo, BCG Copenhagen, BCG Moreau, or BCGMoscow.

In a preferred embodiment, the strain is BCG₁₃₃₁. In addition, the rBCGmay be further genetically engineered to possess other traits, forexample: a perfringolysin O (pfo) gene (in order to facilitate escapefrom the endosome); to express various selection markers such asantibiotic resistance or an auxotrophic selection marker in which a genecritical to the rBCG (e.g. for leucine or lysine synthesis) is deletedand must be complemented (e.g. by an extrachromsomal element thatencodes the missing crucial gene) in order for the bacterium to survive;by deletion of genes or inhibition of the function of gene productswhich suppress apoptosis, etc.

In general, the rBCG of the invention is genetically engineered tooverexpress selected antigens by introduction of genes encoding theantigens of interest into the chromosome of the rBCG under thetranscriptional control of highly active expression control sequences,which may include those which are most active during mammalianinfection. However, the genes encoding the antigens of interest couldalso be expressed from an extrachromosomal plasmid under thetranscriptional control of highly active expression control sequences.Expression control sequences include but are not limited to promoters,ribosomal entry sites, etc.

The present invention further provides compositions for use in elicitingan immune response in and/or vaccinating a mammal. The compositions maybe utilized as a vaccine against Mtb. The compositions of the inventioninclude genetically engineered rBCG as described herein, and apharmacologically suitable carrier. The preparation of such compositionsfor use as vaccines is well known to those of skill in the art.Typically, such compositions are prepared either as liquid solutions orsuspensions, however solid forms such as tablets, pills, powders and thelike are also contemplated. Solid forms suitable for solution in, orsuspension in, liquids prior to administration may also be prepared. Thepreparation may also be emulsified. The active ingredients may be mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredients. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol and the like, or combinationsthereof. In addition, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and the like. In addition, the composition may contain otheradjuvants. If it is desired to administer an oral form of thecomposition, various thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders and the like may be added. The composition ofthe present invention may contain any such additional ingredients so asto provide the composition in a form suitable for administration. Thefinal amount of rBCG in the formulations may vary. However, in general,the amount in the formulations will be from about 1-99%. Thecompositions may further comprise additional adjuvants, suitableexamples of which include but are not limited to Seppic, Quil A,Alhydrogel, etc.

Vaccine formulation also involves studies to determine maximum bacterialviability and stability throughout the manufacturing process. Thisincludes determination of maximum organism viability (live to dead)during culture utilizing a variety of commonly used medium for theculture of Mycobacteria to include the addition of glycerol, sugars,amino acids, and detergents or salts. After culture cells are harvestedby centrifugation or tangential flow filtration and resuspended in astabilizing medium that allows for protection of cells during freezingor freeze-drying process. Commonly used stabilizing agents includesodium glutamate, amino acids or amino acid derivatives, glycerol,sugars or commonly used salts. The final formulation will providesufficient viable organisms to be delivered by intradermal, percutaneousinjection, perfusion or oral delivery with sufficient stability tomaintain and adequate shelf life for distribution and use.

The methods of the present invention involve administering a compositioncomprising the rBCG of the invention in a pharmacologically acceptablecarrier to a subject, usually a human mammal. The vaccine preparationsof the present invention may be administered by any of the many suitablemeans which are well known to those of skill in the art, including butnot limited to by injection, orally, intranasally, by ingestion of afood product containing the antigen, etc. However, in a preferredembodiment, the mode of administration is intradermal injection. Inaddition, the compositions may be administered alone or in combinationwith other medicaments or immunogenic compositions, e.g. as part of amulti-component vaccine. Further, administration may be a single event,or multiple booster doses may be administered at various timed intervalsto augment the immune response. In one embodiment, the vaccinepreparation of the invention is used for an initial immunization, andthe individual receiving the initial vaccine is then “boosted” with oneor more different vaccine compositions, e.g. a known attenuated BCGvaccine. Alternatively, an individual may be vaccinated with anothervaccine preparation and boosted one or more times with the vaccinepreparation of the present invention. In some embodiments of theinvention, the boosting compositions include nucleic acids encoding oneor more Mtb antigens, for example: i) one or more antigens such asRv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c,Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c,Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c,Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c, Rv1886,Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c, Rv1174c,Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132; ii) one or more antigenssuch as Rv1996, Rv2005, Rv2029, Rv2623, Rv2626 and Rv2727; iii) one ormore antigens such as Rv2626, Rv1738, Rv2623, Rv1733, Rv2032, Rv3131,Rv3127, Rv3130c, Rv3804c and Rv1886c; and iv) one or more antigensincluding at least one of a dosR antigen, a reactivation antigen and/ora resuscitation antigen e.g., one antigen from each stage (latency,reactivation and resuscitation) of the life cycle of M. tuberculosis.

A particular advantage of the vaccine preparation of the presentinvention is that administration may be prophylactic, i.e. beforeexposure to the bacteria has occurred, or is suspected to have occurred,or after the fact, i.e. after a known or suspected exposure, ortherapeutically, e.g. after the occurrence of disease symptomsassociated with bacterial infection. This is because antigens that areinvolved in Mtb life cycle or infection and disease processes such asresuscitation and reactivation are included in the vaccine. Thus, thevaccine is useful not only for preventing the initial establishment ofan Mtb infection, but also for preventing the reactivation of a latentMtb infection.

Prior to administration to humans as a vaccine, the geneticallyengineered rBCG strains of the present are tested according to methodsthat are well-known to those of skill in the art. For example, tests fortoxicity, virulence, safety, etc. are carried out in suitable animalmodels, e.g. in mice, guinea pigs, etc., some of which areimmunocompromised. The ability of the vaccine preparations to elicit animmune response is likewise typically tested in suitable animal models,e.g. mice, guinea pigs, etc. In addition, protection studies involvingvaccination, boosting, and subsequent challenge with live Mtb may becarried out using suitable animal models such as mice, guinea pigs, andnon-human primates. Finally, those of skill in the art are familiar withthe arrangements for carrying out clinical trials in consenting humans,in order to test the efficacy of the vaccine preparations. For details,see, for example, United States patent application 20060121054 (Sun etal.) published Jun. 8, 2006, and references cited therein.

By “eliciting an immune response” we mean that administration of thevaccine preparation of the present invention causes the synthesis ofspecific antibodies (at a titer in the range of 1 to 1×10⁶, preferably1×10³, more preferable in the range of about 1×10³ to about 1×10⁶, andmost preferably greater than 1×10⁶) and/or cellular proliferation, asmeasured, e.g. via cellular assays in which IFN-γ production isassessed, for example, by ³H thymidine incorporation, or other suitablemeans. In a preferred embodiment, the immune response is a protectiveimmune response, i.e. the immune response protects the vaccinatedindividual from future challenge with Mtb. However, those of skill inthe art will recognize that a vaccine or immunostimulating preparationneed not provide total protection in order to convey some benefit to apatient. For example, the preparations may elicit immune responses thatslow or lessen the progress of disease symptoms, without fullyeradicating them.

The following non-limiting Examples serve to illustrate the practice ofthis invention.

EXAMPLES Example 1 Selection of Suitable Antigens and Design of rBCGBased on this Selection

An initial group of Mtb proteins (189 antigens, see Table 1) wasselected from all possible 3989 Mtb ORFs, according to the followingselection procedure:(1) Compilation of available data for all 3989 TB ORF products:Literature scan for global analyses (e.g. up/down regulation ofexpression under various conditions such as hypoxia, dormancy,interaction with macrophage, location and persistence in lung tissue;iron regulation; transcriptomics and proteomics profiles; mutationleading to attenuation of virulence; potential high immune responseetc.).(2) Establishing a subset of genes by cross matching the accumulateddata: Selection of antigens that demonstrate positive evidence in anytwo independent studies originating from different criteria mentionedabove.(3) Iterative trimming of the above subset, aiming at selecting aninitial group of candidates (Table 1, 189 antigens), based on the extentof effect documented in the individual studies.(4) Conducting bioinformatic studies for:

-   -   a) Further characterization of the initial group of 189        candidates by domain analyses, prediction of cellular        localization, inspection of genome context, identification of        repeat proteins, and assignment of putative function for the        unknown/hypothetical Ag (sequence similarity searches by blast        against the nr database, NCBI, domain/motifs databases, and        Mtb-related servers and databases: TB-sgc—The TB Structural        Genomics Consortium (Web site located at        www.doe-mbi.ucla.edu/TB/), Tuberculist—the database on        Mycobacterium tuberculosis genetics (Web site located at        genolist.pasteur.fr/TubercuList/), TBDB—an integrated platform        for TB drug discovery (Web site located at www.tbdb.org)        MTBreg—The Database of Conditionally Regulated Proteins in        Mycobacterium tuberculosis (Web site located at        www.doe-mbi.ucla.edu/Services/MTBreg/) and the BioHealthBase        server (Web site located at www.biohealthbase.org/GSearch).    -   b) Immunoinformatic analysis for prediction of human leukocyte        antigen (HLA)-binding targets (by NetCTL) and experimentally        documented T-cell epitopes by database (IEDB) and literature        searches. Implementing various measures for the determination of        epitopes as putative strong binders, and for the selection of        most potent candidates according to the predictions (i.e.        thresholds, and number of supertypes/population coverage).        (5) In depth literature search for data pertaining to aspects of        virulence and vaccine development, for each of the 189 antigens        (Table 1) and/or their orthologs.        (6) Setting up a knowledge dataset of the 189 candidates (Table        1), by integrating all literature and data analyses (steps 4&5        above).        The selected antigens in the initial group presented in Table 1,        include known vaccine candidates, and proteins known to be:        involved in various stages of the life cycle of Mtb (dormancy,        reactivation, resuscitation); tissue specific antigens; antigens        affected by starvation; and, antigens originating in genomic        sequences present in a virulent Mtb strain.

The 189 candidate antigens were ranked in order of likely importance bythe following 14 analyses:

1) macrophage survival/persistence;2) up-regulation of expression by the two-component system MprAB;3) response to hypoxia;4) involvement in reactivation;5) involvement in dormancy;6) expression in lung tissue;7) co-regulation with Rv2031 (Acr);8) whether or not the protein is secreted;9) the ability to act as a B-cell immunogen;10) iron-regulated genes;11) cell wall association or cell wall biogenesis;12) existing vaccine efficacy data;13) the presence of repeat domains;14) T-cell response, determined by a) known experimental evidence (e.g.from the literature) and b) a determination that the antigen likelycontains one or more T-cell epitopes as assessed by theimmunoinformatics program of the Technical University of Denmark(cbs.dtu.dk/services/NetCTL);

The presence or absence of each of these traits was scored for each ofthe 189 genes and a qualitative score was determined and employed as ameasure to rank the list of 189 antigens and choose for the 45 besthits.

The 45 candidates were then further ranked using the same 13 criteria,by assigning internal numerical scores to each of the criteria,according to the intensity of the results and/or relevance to vaccinedevelopment. A list of the top-ranking 45 antigens is given in Table 2,along with their subgrouping according to the class/phase of infection(latency/dormancy, resuscitation/reactivation, classical and others);within the classes, the antigens are sorted by their scores. Table 3presents the 45 antigens prioritization into 3 subset groups accordingto their quantitative, and subsequently—qualitative scores.

Based on this analysis, final selections of groups of antigens for usein the rBCGs were made, usually based on the antigens with the highestoverall scores. In addition, for the final selection, antigens weregrouped according to “type” in that the rBCG includes at least

1) one or more Mtb antigens, including the so-called “classical” Mtbantigens such as 85A, 85B and TB 10.4; and2) at least one Mtb resuscitation/reactivation antigen selected fromRv0867c, Rv1009, Rv1884c, Rv2389c, Rv2450c, Rv0867c, Rv0288, Rv1009,Rv0685, Rv0824c, Rv1349, Rv2744c, Rv3347c, Rv1130, and Rv1169c.

In addition, antigens were selected based on other criteria such asdemonstrated protective efficacy in an animal model, the expression ofthe antigen by Mtb but not BCG, or diminished expression of the antigenin BCG (J Mattow et al., Electrophoresis, 22:2936-2946, 2001, P. R.Jungblut, Molecular Microbiology, 33:1103-1117, 1999, H. J. Mollenkopfet al, Infection and Immunity, 72:6471-6479). Such Mtb-specific antigensinclude Rv1511, Rv2520c, Rv3407, Rv2802c and Rv3710.

Preferred combinations of antigens to be expressed in an rBCG includethe following:

1) Classical antigens Rv1886c, Rv3804c;2) Resuscitation and Reactivation antigens Rv0867c, Rv1884c, Rv2389c;and3) Mtb-specific antigen Rv3407.

Example 2 Construction of a Recombinant BCG Genetically Engineered toExpress at Least One Classical Mtb Antigen, at Least One MtbResuscitation/Reactivation Antigen and in which the DosR Regulon isUpregulated Materials and Methods: General

For the construction of an rBCG described in the following sections,restriction endonucleases (herein “REs”); New England Biolabs Beverly,Mass.), T4 DNA ligase (New England Biolabs, Beverly, Mass.) and Taqpolymerase (Invitrogen, Carlsbad, Calif.) were used according to themanufacturers' protocols; Plasmid DNA was prepared using small-scale(Qiagen MiniprepR kit, Santa Clara, Calif.) or large-scale (QiagenMaxiprepR kit, Santa Clara, Calif.) plasmids DNA purification kitsaccording to the manufacturer's protocols (Qiagen, Santa Clara, Calif.);Nuclease-free, molecular biology grade Milli-Q water, Tris-HCl (pH 7.5),EDTA pH 8.0, 1M MgCl⁻², 100% (v/v) ethanol, ultra-pure agarose, andagarose gel electrophoresis buffer were purchased from Invitrogen,Carlsbad, Calif. RE digestions, PCRs, DNA ligation reactions and agarosegel electrophoresis were conducted according to well-known procedures(Sambrook, et al., Molecular Cloning: A Laboratory Manual. 1, 2, 3;1989); (Straus, et al., Proc Natl Acad Sci USA. March; 87(5): 1889-93;1990). Nucleotide sequencing to verify the DNA sequence of eachrecombinant plasmid described in the following sections was accomplishedby conventional automated DNA sequencing techniques using an AppliedBiosystems automated sequencer, model 373A.

PCR primers were purchased from commercial vendors such as Sigma (St.Louis, Mo.) or synthesized using an Applied Biosystems DNA synthesizer(model 373A). PCR primers were used at a concentration of 150-250 μM andannealing temperatures for the PCR reactions were determined using Clonemanager software version 4.1 (Scientific and Educational Software Inc.,Durham, N.C.). PCRs were conducted in a BioRad thermocycler (BioRad,Hercules, Calif.). The PCR primers for the amplifications were designedusing Clone Manager® software version 4.1 (Scientific and EducationalSoftware Inc., Durham N.C.). The RE digestions and the PCRs weresubsequently analyzed by agarose gel electrophoresis using standardprocedures (Straus et al, supra 1990; and Sambrook et al., supra 1989).A positive clone is defined as one that displays the appropriate REpattern and/or PCR pattern. Plasmids identified through this procedurewere further evaluated using standard DNA sequencing procedures, asdescribed above.

Escherichia coli strains, such as DH5α and Stable2^(R), were purchasedfrom Invitrogen (Carlsbad, Calif.) and served as initial host of therecombinant plasmids. Recombinant plasmids were introduced into E. colistrains by electroporation using a high-voltage eletropulse device, suchas the Gene Pulser (BioRad Laboratories, Hercules, Calif.), set at100-200≠, 15-25 μF and 1.0-2.5 kV, as described (Straus et al, supra1990). Optimal electroporation conditions were identified by determiningsettings that resulted in maximum transformation rates per mcg DNA perbacterium.

E. coli strains are typically grown on tryptic soy agar (Difco, Detroit,Mich.) or in tryptic soy broth (Difco, Detroit, Mich.), which was madeaccording to the manufacturer's directions. Unless stated otherwise, allbacteria were grown at 37° C. in 5% (v/v) CO₂ with gentle agitation.When appropriate, the media was supplemented with antibiotics (Sigma,St. Louis, Mo.). Bacterial strains were typically stored at −80° C.suspended in (Difco) containing 30% (v/v) glycerol (Sigma, St. Louis,Mo.) at ca. 10⁹ colony-forming units (herein referred to as “cfu”) perml.

Mycobacterial strains were cultured in liquid media, such as Middlebrook7H9 or Saulton Synthetic Medium, preferably at 37° C. The strains can bemaintained as static or agitated cultures. In addition, the growth rateof BCG can be enhanced by the addition of oleic acid (0.06% v/v;Research Diagnostics Cat. No. 01257) and detergents such as Tyloxapol(0.05% v/v; Research Diagnostics Cat. No. 70400). The purity of BCGcultures can be evaluated by evenly spreading 100 mcl aliquots of theBCG culture serially diluted (e.g. 10-fold steps from Neat—10⁻⁸) inphosphate buffered saline (herein referred to PBS) onto 3.5 inch platescontaining 25-30 ml of solid media, such as Middlebrook 7H10. Inaddition, the purity of the culture can be further assessed usingcommercially available medium such as thioglycolate medium (Science Lab,catalogue number 1891) and soybean-casein medium (BD, catalogue number211768).

In order to insert the desired antigen expression cassettes into thechromosome of the perfringolysin expressing BCG1331 derivative describedelsewhere, a plasmid was designed in silico and synthesized by DNA2.0(Menlo Park, Calif.). The salient features of this vector (pJFINT)include an E. coli colE1 origin of replication, 3 multiple cloning sitesseparated by transcriptional terminators rmBT1, T2 of pEX18gm, and rnhA,the attP phage integration region of bacteriophage L5 and the integrasegene of bacteriophage L5 (GenBank #Z18946). Immediately upstream of theL5 sequence, a selectable marker cassette consisting of a kanamycinresistance allele aphA from Tn10 (GenBank #AAM97345) and a sacB gene(Genbank # NP_(—)391325) were included. This marker cassette was flankedby direct repeats of the γΔ resolvase binding site from transposonTn1000. This plasmid is incapable of replication in mycobacterialspecies and the L5 attP sequence allows for high frequency recombinationwith the attB region of mycobacterial chromosomes to facilitateintegration of the plasmid sequence into the chromosome. The markercassette can then be removed from the chromosome of the integrant by theintroduction of γΔ resolvase and selection of markerless strains onsolid media containing 10% sucrose.

An antigen expression cassette was designed in silico to encode Rv0867c,Rv1884c, and Rv2389c separated by optimized ribosomal sites under thetranscriptional control of the hsp60 promoter of Mycobacterium bovis andsynthesized by DNA2.0 (Carlsbad Calif.). A second expression cassettewas similarly designed and constructed to encode Rv1886c, Rv3804c andRv3407c under transcriptional control of the hsp60 promoter. Finally, athird cassette encoding Rv3133c (DosR) under the transcriptional controlof the hsp60 promoter was designed and constructed. These three antigenexpression cassettes were ligated into pJFINT such that each wasseparated by a transcriptional terminator (FIG. 2). The resultingplasmid, pRC108, was electroporated into E. coli Stable2 and the plasmidsequence of a kanamycin resistant clone was verified.

The pRC108 plasmid was isolated from a 100 ml E. coli culture andelectroporated into a pfo expressing derivative of BCG Danish 1331.After electroporation, the cells were cultured overnight in 7H9 mediumwith 10% (v/v) OADC and 0.05% (v/v) of Tyloxapol supplementation andplated on 7H10 agar containing 50 ug/ml of kanamycin. As the plasmiddoes not encode a mycobacterial origin of replication, kanamycinresistance in all colonies tested was conferred by integration of theplasmid into the attB site of the BCG genome. Individual colonies werepicked for PCR analysis and inoculated into 7H9 medium with 10% (v/v)OADC and 0.05% (v/v) tyloxapol for analysis of antigen expression. PCRcharacterization of the kanamycin resistant colonies demonstrated thepresence of the entire plasmid sequence in the chromosome of therecombinant BCG, designated AFV-102pRC108. AFV-102pRC108 cultures werewashed with 7H9 and used to inoculate protein-free 7H9tyloxapolcultures. Supernatants of the AFV-102pRC108 cultures were harvested bycentrifugation and immunoblotted with rabbit polyclonal antisera to thetransglycolase domain of the Rpf protein. Rv1009, which is crossreactive with the transglycosylase domains of all Rpf's or rabbitpolyclonal antisera raised against peptides of Rv0867c. Thisdemonstrated the enhanced production of Rv0867c, Rv1884c, and Rv2389cabove the background of the BCG homologs of these proteins (FIG. 3A).This supernatant was similarly immunoblotted with rabbit polyclonalantisera to Ag85complex and the overexpression of Rv1886c and Rv3804cwas observed (FIG. 3B). The cell pellet was immunoblotted with rabbitpolyclonal antisera to Rv3407 and the expression of Rv3407, the homologof which is normally silent in BCG, was confirmed (FIG. 3C). Cellpellets were also immunoblotted with DosR-specific rabbit polyclonalantisera to demonstrate the overexpression of DosR itself and withrabbit antisera to Rv1733c to demonstrate the upregulation of a DosRregulated protein (FIG. 3D).

In order to complete the construction of this vaccine to make itsuitable for human use, the marker cassette of the integrated plasmidwas then removed. Electrocompetent AFV-102pRC108 cells wereelectroporated with plasmid pYUB870hyg, which encodes the γΔ resolvaseof Tn1000, a sacB allele, and a hygromycin resistance gene (GenBank#ABD64366). Transformants resistant to both kanamycin and hygromycinwere selected on 7H10 media and inoculated into 7H9 liquid media with10% (v/v) OADC and 0.05% (v/v) tyloxapol and no antibiotics. After sevendays growth, dilutions of these liquid cultures were plated on 7H10containing 10% sucrose to select for recombinants from which theaphA-sacB marker has been excised and the pYUB870hyg plasmid has beenlost by dilution and selection against the sacB allele.

Sucrose-resistant transformants were picked for PCR analysis of theintegrated antigen cassettes and were inoculated into 7H9 liquid mediafor immunoblot analysis as before. PCR analysis revealed that theantigen expression cassettes were still present in the chromosome andthat the hygromycin resistance marker and sacB gene had been excised.Immunoblotting of supernatants and cell pellets with antisera to Rpf,Ag85 complex, Rv3407, and DosR confirmed that excision of the markercassette from the chromosome did not effect the expression of theinserted antigen cassettes.

Strain AERAS-407 was constructed by removal of the anti-bioticresistance marker from AFV-102pRC108.

Example 3 Demonstration of the Immunogenicity and Protection Elicited byVaccination with AERAS-407 in Mice

To illustrate the immune responses elicited by AERAS-407 and todemonstrate its protective efficacy against tuberculosis, four groups of10 C57/BL6 mice are vaccinated subcutaneously with either 1) saline, 2)BCG, 3) BCG-PfoA or 4) AERAS-407. Mice in groups 2, 3, and 4 receive5×10⁵ cfu of BCG or recombinant BCG. After 10 weeks, five mice from eachgroup are sacrificed for immune assays and the remaining animals arechallenged with 100 cfu aerosolized M. tuberculosis Erdman.

To measure humoral immune responses, a peptide microarray chip has beendesigned. The chip is spotted with overlapping 50 amino acid peptidesgenerated from the sequences of Rv1886c, Rv3804c, Rv3407c, Rv0867c,Rv1884c, Rv2389c, Rv3133c, and all BCG encoded DosR-regulated proteinswhich are at least 2 fold induced by the overexpression of DosR (i.e.DosR regulated genes for which transcription is at least 2 fold greaterin BCG constitutively expressing the DosR regulon than the wild-typeparent strains under aerobic conditions). Pooled sera from each group isincubated with 3 peptide chips for 1 hr at 37° C. The chips are thenwashed with phosphate-buffered saline (PBS) pH7.2 and incubated withfluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG sera(Abcam, Cambridge, Mass.) for 1 hour at 37° C. The chips are then washedagain with PBS and the immunofluorescence is read with a Genepix 4000BArray Scanner (Molecular Devices, Sunnyvale, Calif.). Values for eachpeptide spot are averaged for the 3 chips in each group. Mice receivingAERAS-407 show greater antibody responses to peptides derived fromRv1886c, Rv3804c, Rv3407c, Rv0867c, Rv1884c, Rv2389c and DosR regulatedproteins than unvaccinated mice or mice vaccinated with BCG or BCG-PfoA.

To measure cellular immune responses elicited by AERAS-407, spleens areharvested from each and homogenized. Splenocyte concentrations areadjusted to 5×10⁵ cells/well in R10 media in multichamber plates and areincubated for 3 days at 37° C. with purified Rv1886c, Rv3804c, Rv3407c,Rv0867c, Rv1884c, or Rv2389c at a concentration of 1 μg/ml. In addition,splenocytes from each mouse are incubated with Rv2623 and Rv3130c asthese proteins are known to be highly upregulated in DosR overexpressingstrains and are known to be potent T cell immunogens. After the 3 dayincubation, supernatants are harvested from the splenocyte cultures andassayed by ELISA for interferon-γ produced in response to antigenstimulation. Control cultures of unstimulated splenocytes or PMA/PHAstimulated splenocytes are included as negative and positive controls,respectively.

To quantify protection afforded by vaccination with AERAS-407, the 5remaining mice from each group are sacrificed 10 weeks after challengewith M. tuberculosis and lungs and spleens are aseptically harvestedfrom each animal. Lungs and spleens are homogenized in PBS and dilutionsare plated on 7H10 agar. After 4 weeks of incubation, the number of M.tuberculosis colonies are enumerated for each animals lungs and spleenand corrected for the dilution factor. This value is the number of livetubercle bacilli present in the lungs and spleen of each animal. BCGvaccination typically results in approximately a 1 log reduction incfu/lung and spleen versus saline vaccination. AERAS-407 vaccinationresults in a greater reduction of Mtb load in the lungs and spleens ofmice.

Example 4 Construction of a Second Recombinant BCG Vaccine GeneticallyEngineered to Over Express Selected Classical M. tb Antigens and theDosR Regulon

A second combination of antigens (TB10.4, Ag85B, Ag85A and Rv3407) wasalso overexpressed in the rBCG AFV102 strain by integration into thechromosome at the attB site. This was carried out in a manner similar tothat described in Example 2 for the Aeras 407 construct. To integratethis second antigen set, the integration plasmid pAF707 (depictedschematically in FIG. 4) was constructed as described for pJFINT inExample 2 except for the following differences: a hygromycin resistancegene was used in place of the kanamycin resistance gene, and the PBlafpromoter was used to drive antigens TB10.4, Ag85B, Ag85A and Rv3407 forover expression in the order listed. All other general materials andmethods were similar to those described in Example 2. The resultantstrain was designated Aeras 406. The final strain was further analyzedby PCR followed by sequencing analysis and confirmed to have the desiredgenotype. The strain was further demonstrated to overexpress theselected antigens by SDS-PAGE analysis and immunoblot testing asdescribed in Example 2 (data not shown).

Example 5 Protection of Non-Human Primates from Tuberculosis byVaccination with AERAS-407

The protective efficacy of AERAS-407, particularly in relation to theenhanced capacity to prevent latent infection and reactivation ofdisease, is best tested in rhesus macaques as mice do not creategranulomatous latent foci as humans and other primates do. To evaluatethe protective efficacy of AERAS-407 in NHP's, four groups of six weightand sex-matched rhesus macaques are vaccinated with 1) saline, 2) BCG1331, 3) BCG-Pfo, or 4) AERAS-407. Each animal in groups 2-4 receives5×10⁵ cfu of the respective BCG or rBCG by intradermal injection.Fifteen weeks after vaccination, all animals are challenged by bronchialinstallation of approximately 300 cfu of M. tuberculosis Erdman. Allanimals are evaluated monthly for six months for clinical symptoms oftuberculosis by chest X-ray, weight, feeding, cough, lethargy, andimmune responses to TB specific proteins. All animals that die duringthe six month observation period are necropsied and tissue pathology andMtb burden by organ is measured as in Example 3. All moribund animalsare humanely euthanized and similarly examined. Six monthspost-challenge all surviving animals are euthanized and necropsied fortissue pathology and Mtb burden in lungs, liver and spleen. AERAS-407vaccination results in decreased mortality, decreased tissue damage andlower counts of viable Mtb organisms in the lungs of experimentallyinfected animals.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-8. (canceled)
 9. A method for immunizing a subject against infectionby Mycobacterium tuberculosis (Mtb) or for eliciting an immune responseto Mtb in said subject, comprising the step of administering to saidsubject a composition comprising a recombinant Bacille Calmette-Guerin(BCG) comprising at least one DosR regulon gene that is up-regulated,and at least two nucleic acid sequences which are different from eachother, each of which encode one or more genes that are overexpressed,said at least two nucleic acid sequences including i) a first nucleicacid sequence encoding at least one Mycobacterium tuberculosis (Mtb)antigen; and ii) a second nucleic acid sequence encoding at least oneMtb reactivation antigen that is not a DosR antigen.
 10. The method ofclaim 9, further comprising the step of administering a boostingcomposition comprising one or more Mtb antigens.
 11. The method of claim10, wherein said one or more antigens is selected from the groupconsisting of Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127,Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006,Rv2029c, Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c,Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c,Rv1886, Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c,Rv1174c, Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132.
 12. The method ofclaim 10, wherein said one or more antigens is selected from the groupconsisting of Rv1996, Rv2005, Rv2029, Rv2623, Rv2626 and Rv2727.
 13. Themethod of claim 10, wherein said one or more antigens is selected fromthe group consisting of Rv2626, Rv1738, Rv2623, Rv1733, Rv2032, Rv3131,Rv3127, Rv3130c, Rv3804c and Rv1886c.
 14. The method of claim 10,wherein said one or more antigens include at least one antigen from eachstage of a life cycle of Mtb.
 15. A method for preventing a recurrenceof symptoms of tuberculosis in a patient with a latent Mycobacteriumtuberculosis (Mtb) infection, comprising the step of administering tosaid patient a composition comprising a recombinant BacilleCalmette-Guerin (BCG) comprising at least one DosR regulon gene that isup-regulated, and at least two nucleic acid sequences which aredifferent from each other, each of which encode one or more genes thatare overexpressed, said at least two nucleic acid sequences including i)a first nucleic acid sequence encoding at least one Mycobacteriumtuberculosis (Mtb) antigen; and ii) a second nucleic acid sequenceencoding at least one Mtb reactivation antigen that is not a DosRantigen.
 16. The method of claim 15, further comprising the step ofadministering a boosting composition comprising nucleic acid sequencesencoding one or more Mtb antigens.
 17. The method of claim 16, whereinsaid one or more antigens is selected from the group consisting ofRv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c,Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c,Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c,Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c, Rv1886,Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c, Rv1174c,Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132.
 18. The method of claim 16,wherein said one or more antigens is selected from the group consistingof Rv1996, Rv2005, Rv2029, Rv2623, Rv2626 and Rv2727.
 19. The method ofclaim 16, wherein said one or more antigens is selected from the groupconsisting of Rv2626, Rv1738, Rv2623, Rv1733, Rv2032, Rv3131, Rv3127,Rv3130c, Rv3804c and Rv1886c.
 20. The method of claim 16, wherein saidone or more antigens include at least one antigen from each stage of alife cycle of Mtb.
 21. (canceled)
 22. The method of claim 9 wherein afirst nucleic acid sequence of said at least two nucleic acid sequenceencodes an antigen selected from the group consisting of Rv2450c,Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744,Rv3347c, Rv1130, Rv1169c; and wherein a second nucleic acid sequence ofsaid at least two nucleic acid sequences encodes an antigen selectedfrom the group consisting of Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c,Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131,Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009,Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c,Rv1130, Rv1169c, Rv1886, Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467,Rv3873, Rv1908c, Rv1174c, Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132.23. The method of claim 15 wherein a first nucleic acid sequence of saidat least two nucleic acid sequence encodes an antigen selected from thegroup consisting of Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288,Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c; and wherein a secondnucleic acid sequence of said at least two nucleic acid sequencesencodes an antigen selected from the group consisting of Rv1738, Rv2623,Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628,Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c,Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685,Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c, Rv1886, Rv1980c, Rv3804c,Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c, Rv1174c, Rv2780, Rv2620c,Rv1793, Rv1349 and Rv3132.